CN115557853A - 一种亮氨酸氨基肽酶荧光探针及其制备方法和应用 - Google Patents
一种亮氨酸氨基肽酶荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种亮氨酸氨基肽酶荧光探针及其制备方法和应用,本发明提供了能快速检测亮氨酸氨基肽酶的发光型荧光探针,简称探针DCM‑LAP;本发明的DCM‑LAP荧光探针与LAP响应后,656nm处的荧光强度增强。本发明DCM‑LAP荧光探针可以通过共聚焦荧光显微镜检测活细胞的LAP,并进行荧光成像。本发明的DCM‑LAP荧光探针合成简单,产率较高,不仅能够对水溶液中LAP进行特异性和快速检测,还可实现肿瘤微环境中(乳腺癌)LAP的检测,具有一定的潜在实用价值。
Description
技术领域
本发明涉及荧光探针技术领域,特别涉及一种亮氨酸氨基肽酶荧光探针及其制备方法和应用。
背景技术
亮氨酸氨基肽酶(LAP)是生物体内一种重要的蛋白水解酶,可以催化蛋白质或肽底物氨基末端的亮氨酸残基水解。LAP广泛存在于哺乳动物、植物、微生物和肿瘤细胞中,参与调控肿瘤细胞增殖、侵袭和耐药性等重要生理病理过程。LAP的异常表达与乳腺癌、肝癌等疾病密切相关,是一种潜在的肿瘤生物标志物。因此,生命体系中LAP的原位动态和选择性监测有助于LAP相关疾病的诊断和治疗。
荧光成像具有时间、空间分辨率高,无创检测等优点。它已广泛应用于化学和生物医学研究,特别是高信噪比的可激活荧光探针。最近,一些LAP激活的荧光探针已经被报道。然而,这些探针在体内检测研究中仍面临波长短、灵敏度有限、选择性低等问题,不利于实时的体内生物成像。因此,具有长波长、高灵敏度、高特异性荧光响应的理想探针对相关疾病的早期检测显得尤为迫切。由于生物组织在近红外(NIR)区域的吸收和自发荧光减少,迫切需要开发近红外荧光成像探针。该类探针的开发,将实现相关疾病(如乳腺癌)的早期精准检测,提高检测分析的准确性和可靠性。
发明内容
本发明提供了一种亮氨酸氨基肽酶荧光探针及其制备方法和应用,其目的是为了解决背景技术存在的上述问题。
为了达到上述目的,本发明的实施例提供了能快速检测亮氨酸氨基肽酶的发光型荧光探针,简称探针DCM-LAP;并进一步提供了探针DCM-LAP的制备方法和应用。
本发明提供了一种亮氨酸氨基肽酶荧光探针,其分子式为C26H32N4O,结构如式I:
进一步的,所述亮氨酸氨基肽酶荧光探针能抗钠离子、钾离子、钙离子、镁离子、氯离子、硫酸根离子、醋酸根离子、碳酸氢根离子、硝酸根离子、过氧化氢、次氯酸、半胱氨酸、谷胱甘肽、硫化氢、一氧化氮、硝基还原酶、碱性磷酸酶、醌氧化还原酶1、氨基肽酶的干扰。
基于一个发明总的构思,本发明还提供了上述的亮氨酸氨基肽酶荧光探针的制备方法,包括如下步骤:
将化合物N-(叔丁氧羰基)-L-新戊基甘氨酸(Boc-beta-t-butyl-L-alanine),O-(7-氮杂苯并三氮唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)和三乙胺(Et3N)用二氯甲烷溶解,常温下搅拌;在氮气保护下加入化合物DCM,常温下搅拌;在真空条件下去除溶剂,获得混合物;将混合物和三氟乙酸溶解在无水二氯甲烷中,在室温下搅拌后静置;在真空条件下去除溶剂,用硅胶柱层析纯化,获得亮氨酸氨基肽酶,即DCM-LAP;
所述DCM-LAP的合成路径如下:
进一步的,所述化合物DCM、HATU、Boc-beta-t-butyl-L-alanine的质量比为55~60:75~78:49~51。
进一步的,所述三乙胺的用量为8μL,无水二氯甲烷的用量为6mL,三氟乙酸的用量为2~7mL。
进一步的,所述硅胶柱层析所用试剂为CH2Cl2/EtOH=10:1。
进一步的,所述去除溶剂采用旋转蒸发仪减压蒸馏法进行去除无水二氯甲烷。
进一步的,所述加入三氟乙酸之后,得到化合物DCM-LAP,用硅胶层析柱纯化。
本发明还提供了上述的亮氨酸氨基肽酶荧光探针或上述的制备方法获得的亮氨酸氨基肽酶荧光探针在检测亮氨酸氨基肽酶中的应用,包括检测水环境中的亮氨酸氨基肽酶、生物样品中的亮氨酸氨基肽酶。
进一步的,所述亮氨酸氨基肽酶的荧光光谱在656nm处出现峰值,荧光光谱仪的激发波长为440nm。
上述应用,具体的,包括:
观察加入亮氨酸氨基肽酶前后待测水环境的荧光光谱的变化,荧光激发波长为440nm;或者,观察加入亮氨酸氨基肽酶荧光探针前后待测生物环境的荧光成像图的变化。
所述生物环境,可以是肿瘤细胞。
所述荧光光谱的变化是指:荧光光谱中,656nm处的荧光峰值的变化;如果656nm处峰值升高,则说明含有LAP。优选地,采用荧光光谱仪观察荧光光谱。
所述荧光成像图的变化是指:将探针母液加入到生物样品中,用激光共聚焦显微镜,使用激发波长为440nm的光源激发,收集红色通道的荧光;观察到红色通道荧光增强,则说明含有LAP。优选地,采用激光共聚焦扫描显微镜。
上述应用,具体的,包括以下步骤:
(1)将探针DCM-LAP溶于二甲基亚砜(DMSO),制成探针母液;
(2)将探针母液加入到待测液中;
用荧光光谱仪测试待测液的荧光光谱,观察其在656nm处的荧光峰值的变化,如果656nm处峰值变大,则说明含有LAP;其中,荧光光谱仪激发波长为440nm;
(3)将探针母液加入到生物样品中,用激光共聚焦显微镜,使用激发波长为440nm的光源激发,收集红色通道的荧光;若观察到红色通道荧光增强,则说明含有LAP。
首先,水溶液中的LAP可以引起荧光探针的荧光光谱变化,即荧光探针与LAP的特异反应引发近红外荧光信号变化,主要是由于荧光探针上特异性识别基团能与分析物LAP结合,具体表现为LAP会水解荧光探针上的识别单元亮氨酸,切断荧光探针的酰胺键导致荧光探针的分子内电荷转移增强,从而使其荧光恢复,实现快速、高灵敏LAP检测。
因此,本发明可以通过观察荧光光谱仪中光谱的变化程度判断溶液中的LAP含量变化;其次,通过共聚焦显微镜对孵育了荧光探针的活细胞进行荧光成像,观察红色通道荧光信号的变化以达到检测生物环境中LAP的目的。
本发明的上述方案具有如下的有益效果:
(1)探针合成简单,并且产率较高;(2)实现了水溶液中LAP的特异性和快速检测;(3)实现了肿瘤环境(如乳腺癌)中LAP的检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例1中DCM-LAP的1HNMR图谱;
图2是本发明实施例1中DCM-LAP的13CNMR图谱;
图3是本发明实施例2中DCM-LAP(5μmol/L)在PBS缓冲溶液(浓度25mmol/L,10%DMSO,pH7.4)随不同量LAP的加入荧光谱图的变化情况;
图4是本发明实施例3中DCM-LAP(5μmol/L)在PBS缓冲溶液(浓度25mmol/L,10%DMSO,pH7.4)对不同干扰分析物的选择性柱状荧光数据图;
图5是本发明实施例4中DCM-LAP(5μmol/L)在PBS缓冲溶液(浓度25mmol/L,10%DMSO,pH7.4)在不同温度下荧光数据图;
图6是本发明实施例5中DCM-LAP(5μmol/L)实时检测HepG2中LAP的共聚焦荧光成像图,加入DCM-LAP(5μmol/L)后在0分钟、5分钟、10分钟、15分钟、20分钟和30分钟进行共聚焦荧光成像。
图7是本发明实施例5中不同浓度bestatin(20μmol/L、50μmol/L、100μmol/L)预处理1小时后,再用DCM-LAP(5μmol/L)检测HepG2中LAP的共聚焦荧光成像图,第一列是加入5μmol/LDCM-LAP孵育30分钟后的成像;第二列是提前用bestatin(20μmol/L)预处理1小时后,再加入5μmol/LDCM-LAP孵育30分钟后的成像;第三列是提前用bestatin(50μmol/L)预处理1小时后,再加入5μmol/LDCM-LAP孵育30分钟后的成像;第四列是提前用bestatin(100μmol/L)预处理1小时后,再加入5μmol/LDCM-LAP孵育30分钟后的成像。
图8是本发明实施例5中DCM-LAP(5μmol/L)检测不同种类肿瘤细胞中LAP含量的共聚焦荧光成像图,A549是加入DCM-LAP(5μmol/L)孵育30分钟后的A549细胞的成像;HeLa是加入DCM-LAP(5μmol/L)孵育30分钟后的HeLa细胞的成像,HepG2是加入DCM-LAP(5μmol/L)孵育30分钟后的HepG2细胞的成像;MCF-7是加入DCM-LAP(5μmol/L)孵育30分钟后的MCF-7细胞的成像。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
除非另有定义,下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的,并不是旨在限制本发明的保护范围。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
本发明针对现有的问题,提供了一种亮氨酸氨基肽酶荧光探针及其制备方法和应用。
根据本发明实施例,本发明提供了一种亮氨酸氨基肽酶,其分子式为C26H32N4O,结构如式I:
进一步的,所述亮氨酸氨基肽酶荧光探针能抗钠离子、钾离子、钙离子、镁离子、氯离子、硫酸根离子、醋酸根离子、碳酸氢根离子、硝酸根离子、过氧化氢、次氯酸、半胱氨酸、谷胱甘肽、硫化氢、一氧化氮、硝基还原酶、碱性磷酸酶、醌氧化还原酶1、氨基肽酶的干扰。
基于一个发明总的构思,本发明还提供了上述的亮氨酸氨基肽酶荧光探针的制备方法,包括如下步骤:
将化合物N-(叔丁氧羰基)-L-新戊基甘氨酸(Boc-beta-t-butyl-L-alanine),O-(7-氮杂苯并三氮唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)和三乙胺(Et3N)用二氯甲烷溶解,常温下搅拌;在氮气保护下加入化合物DCM,常温下搅拌;在真空条件下去除溶剂,获得混合物;将混合物和三氟乙酸溶解在无水二氯甲烷中,在室温下搅拌后静置;在真空条件下去除溶剂,用硅胶柱层析纯化,获得亮氨酸氨基肽酶,即DCM-LAP;
所述DCM-LAP的合成路径如下:
进一步的,所述化合物DCM、HATU、Boc-beta-t-butyl-L-alanine的质量比为55~60:75~78:49~51。
进一步的,所述三乙胺的用量为8μL,无水二氯甲烷的用量为6mL,三氟乙酸的用量为2~7mL。
进一步的,所述硅胶柱层析所用试剂为CH2Cl2/EtOH=10:1。
进一步的,所述去除溶剂采用旋转蒸发仪减压蒸馏法进行去除无水二氯甲烷。
进一步的,所述加入化合物三氟乙酸,得到化合物DCM-LAP,用硅胶层析柱纯化。
本发明还提供了上述的亮氨酸氨基肽酶荧光探针或上述的制备方法获得的亮氨酸氨基肽酶荧光探针在检测亮氨酸氨基肽酶中的应用,包括检测水环境中的亮氨酸氨基肽酶、生物样品中的亮氨酸氨基肽酶。
进一步的,所述亮氨酸氨基肽酶荧光探针的荧光光谱在656nm处出现峰值,荧光光谱仪的激发波长为440nm。
上述应用,具体的,包括:
观察加入亮氨酸氨基肽酶前后待测水环境的荧光光谱的变化,荧光激发波长为440nm;或者,观察加入亮氨酸氨基肽酶荧光探针前后待测生物环境的荧光成像图的变化。
所述生物环境,可以是活肿瘤细胞。
所述荧光光谱的变化是指:荧光光谱中,656nm处的荧光峰值的变化;如果656nm处峰值升高,则说明含有LAP。优选地,采用荧光光谱仪观察荧光光谱。
所述荧光成像图的变化是指:将探针母液加入到生物样品中,用激光共聚焦显微镜,使用激发波长为440nm的光源激发,收集红色通道的荧光;观察到红色通道荧光增强,则说明含有LAP。优选地,采用激光共聚焦扫描显微镜。
上述应用,具体的,包括以下步骤:
(1)将探针DCM-LAP溶于二甲基亚砜(DMSO),制成探针母液;
(2)将探针母液加入到待测液中;
用荧光光谱仪测试待测液的荧光光谱,观察期在656nm处的荧光峰值的变化,如果656nm处峰值变大,则说明含有LAP;其中,荧光光谱仪激发波长为440nm;
(3)将探针母液加入到生物样品中,用激光共聚焦显微镜,使用激发波长为440nm的光源激发,收集红色通道的荧光;若观察到红色通道荧光增强,则说明含有LAP。
首先,水溶液中的LAP可以引起荧光探针的荧光光谱变化,即荧光探针与LAP的特异反应引发近红外荧光信号变化,主要是由于荧光探针上特异性识别基团能与分析物LAP结合,具体表现为LAP会水解荧光探针上的识别单元亮氨酸,切断荧光探针的酰胺键导致荧光探针的分子内电荷转移增强,从而使其荧光恢复,实现快速、高灵敏、高选择的LAP检测。
实施例1
DCM-LAP的合成:
首先将化合物N-(叔丁氧羰基)-L-新戊基甘氨酸(Boc-beta-t-butyl-L-alanine)(49.0mg,0.2mmol),O-(7-氮杂苯并三氮唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸酯(76.0mg,0.2mmol)和三乙胺(8μL)置于圆底烧瓶,加入6mL干燥的二氯甲烷溶液,搅拌该混合液30分钟,然后将化合物DCM(57.8mg,0.2mmol)加入以上反应体系,常温下搅拌6小时。然后在真空条件下除去溶剂,得到混合物。然后将混合物和三氟乙酸(2mL)溶解在3mL无水二氯甲烷中。将混合物在室温下搅拌8小时。然后在真空条件下去除溶剂,用硅胶柱层析纯化,得到黄色固体DCM-LAP(25.6mg,产率31%)。其氢谱和碳谱如图1、2所示,1H NMR(400MHz,CD3OD)δ7.69(d,J=8.7Hz,2H),7.62(d,J=8.7Hz,2H),7.23(d,J=16.1Hz,1H),7.13(d,J=16.2Hz,1H),6.85(s,1H),3.67(m,1H),2.63(s,2H),2.57(s,2H),2.00(m,1H),1.53(m,1H),1.10(s,6H),1.03(s,9H).13C NMR(100MHz,CD3OD)δ173.2,169.8,155.2,139.4,136.7,132.2,128.2,128.1,122.6,119.9,113.2,112.5,77.1,52.8,42.5,38.4,31.5,29.7,29.4,28.8,28.7,26.7,22.4.HRMS(ESI)C26H32N4O-[M-H]-,calculated 415.2498,found415.2498.
实施例2
DCM-LAP与不同当量LAP反应的荧光光谱变化
取实施例1制备的DCM-LAP溶于DMSO中,制成浓度为500μmol/L探针母液。用25mmol/L PBS(10%DMSO,pH7.4)稀释至5μmol/L作为测试溶液,加入不同浓度的LAP母液,配置成探针浓度为5μmol/L的PBS测试溶液。用荧光光谱仪测试探针与不同浓度LAP反应液的荧光光谱变化(激发波长为440nm),荧光光谱变化情况如图3所示。随着加入LAP浓度的逐渐增加,探针在656nm处的荧光峰值逐渐增强。
实施例3
DCM-LAP对不同干扰分析物的选择性研究
从实施例2中荧光探针母液中取出部分用25mmol/L PBS(pH7.4)稀释至5μmol/L作为测试溶液,分别加入不同分析物:钠离子、钾离子、钙离子、镁离子、氯离子、硫酸根离子、醋酸根离子、碳酸氢根离子、硝酸根离子、过氧化氢、次氯酸、半胱氨酸、谷胱甘肽、硫化氢、一氧化氮、硝基还原酶、碱性磷酸酶、醌氧化还原酶1、氨基肽酶。反应90分钟后检测测试液的荧光光谱变化。如由图4所示,可以发现,相对于空白测试液,各种分析物的测试液荧光强度均没有明显变化。然而,加入LAP的测试液的荧光强度发生了显著增强。实验结果说明DCM-LAP对LAP具有良好的选择性。
实施例4
DCM-LAP在不同温度下对LAP的响应研究
取实施例1制备的DCM-LAP溶于DMSO中,制成浓度为500μmol/L探针母液。用25mmol/L PBS(10%DMSO,pH 7.4)稀释至5μmol/L作为测试溶液,加入不同浓度的LAP母液,配置成探针浓度为5μmol/L的PBS测试溶液。用荧光光谱仪测试探针与LAP在不同温度下(25,37,40摄氏度)的荧光光谱变化(激发波长为440nm),荧光光谱变化情况如图5所示。在生理温度37摄氏度条件下,探针在656nm处的荧光峰值较强。
实施例5
DCM-LAP与细胞中LAP的荧光成像研究
从实施例2中荧光探针母液中取出10μL加入到育有HepG2细胞的培养皿(含高糖DMEM培养基添加10%胎牛血清和1%抗生素)中,探针浓度为5μmol/L;实验组样品使用共聚焦显微镜检测红色通道荧光强度,每隔5分钟成像一次,使用激发波长为440nm的光源激发,收集红色通道的荧光如图6所示。可以看到实验组红色荧光逐渐增强。实验结果说明DCM-LAP可以很好地,快速地识别肿瘤细胞中的LAP,具有潜在的实际应用价值。
从实施例2中荧光探针母液中取出10μL加入到育有HepG2细胞的培养皿(含高糖DMEM培养基添加10%胎牛血清和1%抗生素)中,探针浓度为5μmol/L,孵育30分钟,作为控制组a;实验组b样品用bestatin(20μmol/L)预处理细胞1小时,随后用探针DCM-LAP(5μmol/L,30分钟)孵育,实验组c用bestatin(50μmol/L)预处理细胞1小时,随后用探针DCM-LAP(5μmol/L,30分钟)孵育,实验组d样品用bestatin(100μmol/L)预处理细胞1小时,随后用探针DCM-LAP(5μmol/L,30分钟)孵育。随后分别用共聚焦显微镜对控制组和实验组进行荧光成像,使用激发波长为440nm的光源激发,收集红色通道的荧光,结果如图7所示。在控制组a的荧光成像中,红色荧光最强烈;在实验组b中,可以观察到较明显减弱的红色通道荧光,在实验组c中能观察到明显减弱的红色荧光,在实验组d能观察到显著减弱的红色荧光。实验结果说明探针DCM-LAP可以通过共聚焦显微镜检测肿瘤细胞环境中的内源性LAP,具有潜在的实际应用价值。
从实施例2中荧光探针母液中分别取出10μL加入到育有A549、Hela、HepG2、MCF-7细胞的培养皿(含高糖DMEM培养基添加10%胎牛血清和1%抗生素)中,探针浓度为5μmol/L,孵育30分钟。随后分别用共聚焦显微镜进行荧光成像,使用激发波长为440nm的光源激发,收集红色通道的荧光,结果如图8所示。在A549细胞的荧光成像中,观察到明显的红色通道荧光;在Hela细胞的荧光成像中,观察到明显的红色通道荧光;在HepG2细胞的荧光成像中,观察到明显的红色通道荧光;在MCF-7细胞的荧光成像中,观察到明显的红色通道荧光。实验结果说明探针DCM-LAP可以通过共聚焦显微镜检测不同种类肿瘤细胞环境中的LAP,具有潜在的实际应用价值。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
2.根据权利要求1所述的亮氨酸氨基肽酶荧光探针,其特征在于,所述亮氨酸氨基肽酶荧光探针能抗钠离子、钾离子、钙离子、镁离子、氯离子、硫酸根离子、醋酸根离子、碳酸氢根离子、硝酸根离子、过氧化氢、次氯酸、半胱氨酸、谷胱甘肽、硫化氢、一氧化氮、硝基还原酶、碱性磷酸酶、醌氧化还原酶1、氨基肽酶的干扰。
3.如权利要求1或2所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,包括如下步骤:
将化合物N-(叔丁氧羰基)-L-新戊基甘氨酸(Boc-beta-t-butyl-L-alanine),O-(7-氮杂苯并三氮唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)和三乙胺(Et3N)用二氯甲烷溶解,常温下搅拌;在氮气保护下加入化合物DCM,常温下搅拌;在真空条件下去除溶剂,获得混合物;将混合物和三氟乙酸溶解在无水二氯甲烷中,在室温下搅拌后静置;在真空条件下去除溶剂,用硅胶柱层析纯化,获得亮氨酸氨基肽酶荧光探针,即DCM-LAP。
4.根据权利要求3所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,所述化合物DCM、HATU、Boc-beta-t-butyl-L-alanine的质量比为55~60:75~78:49~51。
5.根据权利要求3所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,所述三乙胺的用量为8μL,无水二氯甲烷的用量为6mL,三氟乙酸的用量为2~7mL。
6.根据权利要求3所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,所述硅胶柱层析所用试剂为CH2Cl2/EtOH=10:1。
7.根据权利要求3所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,所述去除溶剂采用旋转蒸发仪减压蒸馏法进行去除二氯甲烷。
8.根据权利要求3所述的亮氨酸氨基肽酶荧光探针的制备方法,其特征在于,所述加入三氟乙酸之后,得到化合物DCM-LAP,用硅胶层析柱纯化。
9.如权利要求1或2所述的亮氨酸氨基肽酶荧光探针或如权利要求3~8任一项所述的制备方法获得的亮氨酸氨基肽酶荧光探针在检测亮氨酸氨基肽酶中的应用,其特征在于,包括检测水环境中的亮氨酸氨基肽酶、生物样品中的亮氨酸氨基肽酶。
10.根据权利要求9所述的应用,其特征在于,所述亮氨酸氨基肽酶的荧光光谱在656nm处出现峰值,荧光光谱仪的激发波长为440nm。
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